Methods have been developed in this laboratory for the isolation of pulse-labeled fibroin messenger RNA from the silkworm Bombyx mori. These methods have permitted the demonstration of putative mRNA precursor molecules which can be observed by pulse labeling at low temperature. To further characterize these putative mRNA precursor molecules we propose to utilize hybridization tools prepared by genetic engineering techniques. DNA obtained from fibroin cDNA clones or genomic fibroin DNA clones will be used for a variety of hybridization assays with Ribonuclease H and S1 nuclease. These assays will beused to construct a linear map of the precursor molecule, and to define the temporal order of processing reactions in vivo. The processing patterns of different allelic forms of the fibroin gene will be compared. Attempts will be made to follow processing reactions in isolated nuclei using an assay based on Ribonuclease H-generated mRNA fragments. The assay requires relatively small amount of fibroin mRNA radioactivity. We will attempt to identify processing activities of insect cell fractions by using labeled mRNA precursor as a substrate for such activities in an in vitro assay. mRNA maturation will also be studied from the point of view of translational capacity of the newly-synthesized molecules. The time at which mRNA precursor molecules become capable of forming initiation complexes with ribosomal subunits will be investigated. This will be compared to in vivo measurements of the time of entry of new mRNA into polysomal structures in vivo.